The present invention relates to a novel member of the TNF-Ligand superfamily. More specifically, isolated nucleic acid molecules are provided encoding a human Apoptosis Inducing Molecule II (AIM II). AIM II polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of AIM II activity. Also provided are therapeutic methods for treating lymphadenopathy, aberrant bone development, autoimmune and other immune system diseases, graft versus host disease, rheumatoid arthritis, osteoarthritis and to inhibit neoplasia, such as tumor cell growth.
Human tumor necrosis factors a (TNF-xcex1) and xcex2 (TNF-xcex2, or lymphotoxin) are related members of a broad class of polypeptide mediators, which includes the interferons, interleukins and growth factors, collectively called cytokines (Beutler, B. and Cerami, A., Annu. Ret. Immunol. 7:625-655 (1989)).
Tumor necrosis factor (TNF-xcex1 and TNF-xcex2) was originally discovered as a result of its anti-tumor activity, however, now it is recognized as a pleiotropic cytokine capable of numerous biological activities including apoptosis of some transformed cell lines, mediation of cell activation and proliferation and also as playing important roles in immune regulation and inflammation.
To date, known members of the TNF-ligand superfamily include TNF-xcex1, TNF-xcex2 (lymphotoxin-xcex1), LT-xcex2, OX40L, Fas ligand, CD30L, CD27L, CD40L and 4-IBBL. The ligands of the TNF ligand superfamily are acidic, TNF-like molecules with approximately 20% sequence homology in the extracellular domains (range, 12%-36%) and exist mainly as membrane-bound forms with the biologically active form being a trimeric/multimeric complex. Soluble forms of the TNF ligand superfamily have only been identified so far for TNF, LTxcex1, and Fas ligand (for a general review, see Gruss, H. and Dower, S. K., Blood, 85(12):3378-3404 (1995)), which is hereby incorporated by reference in its entirety.
These proteins are involved in regulation of cell proliferation, activation, and differentiation, including control of cell survival or death by apoptosis or cytotoxicity (Armitage, R. J., Curr. Opin. Immunol. 6:407 (1994) and Smith, C. A., Cell 75:959 (1994)).
Mammalian development is dependent on both the proliferation and differentiation of cells as well as programmed cell death which occurs through apoptosis (Walker et al., Methods Achiev. Exp. Pathol. 13:18 (1988). Apoptosis plays a critical role in the destruction of immune thymocytes that recognize self antigens. Failure of this normal elimination process may play a role in autoimmune diseases (Gammon et al., Immunology Today 12:193 (1991)).
Itoh et al. (Cell 66:233(1991)) described a cell surface antigen, Fas/CD95 that mediates apoptosis and is involved in clonal deletion of T-cells. Fas is expressed in activated T-cells, B-cells, neutrophils and in thymus, liver, heart and lung and ovary in adult mice (Watanabe-Fukunaga et al., J. Immunology. 148: 1274 (1992)). In experiments where a monoclonal Ab to Fas is cross-linked to Fas, apoptosis is induced (Yonehara et al., J. Exp. Med. 169:1747 (1989); Trauth et al., Science 245:301 (1989)). In addition, there is an example where binding of a monoclonal Ab to Fas may stimulate T-cells under certain conditions (Alderson et al., J. Exp. Med. 178:2231 (1993)).
Fas antigen is a cell surface protein of relative MW of 45 Kd. Both human and murine genes for Fas have been cloned by Watanabe-Fukunaga et al., (J. Immunol. 148:1274 (1992)) and Itoh et al. (Cell 66:233 (1991)). The proteins encoded by these genes are both transmembrane proteins with structural homology to the Nerve Growth Factor/Tumor Necrosis Factor receptor superfamily, which includes two TNF receptors, the low affinity Nerve Growth Factor receptor and the LTxcex2 receptor CD40, CD27, CD30, and OX40.
Recently the Fas ligand has been described (Suda et al., Cell 75:1169 (1993)). The amino acid sequence indicates that Fas ligand is a type II transmembrane protein belonging to the TNF family. Fas ligand is expressed in splenocytes and thymocytes. The purified Fas ligand has a MW of 40 kd.
Recently, it has been demonstrated that Fas/Fas ligand interactions are required for apoptosis following the activation of T-cells (Ju et al., Nature 373:444 (1995); Brunner et al., Nature 373:441 (1995)). Activation of T-cells induces both proteins on the cell surface. Subsequent interaction between the ligand and receptor results in apoptosis of the cells. This supports the possible regulatory role for apoptosis induced by Fas/Fas ligand interaction during normal immune responses.
The polypeptide of the present invention has been identified as a novel member of the TNF ligand super-family based on structural and biological similarities.
Clearly, there is a need for factors that regulate activation, and differentiation of normal and abnormal cells. There is a need, therefore, for identification and characterization of such factors that modulate activation and differentiation of cells, both normally and in disease states. In particular, there is a need to isolate and characterize additional Fas ligands that control apoptosis for the treatment of autoimmune disease, graft versus host disease, rheumatoid arthritis and lymphadenopathy.
The present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding the AIM II polypeptide having the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA deposited as ATCC Deposit Number 97689 on Aug. 22, 1996. The present invention also provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding the AIM II polypeptide having the amino acid sequence shown in FIGS. 1C and 1D (SEQ ID NO:39) or the amino acid sequence encoded by the cDNA deposited as ATCC Deposit Number 97483 on Mar. 15, 1996.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of AIM II polypeptides or peptides by recombinant techniques.
The invention further provides an isolated AIM II polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
As used herein the term xe2x80x9cAIM IIxe2x80x9d polypeptide includes membrane-bound proteins (comprising, or alternatively consisting of, a cytoplasmic domain, a transmembrane domain, and an extracellular domain) as well as truncated proteins that retain the AIM II polypeptide activity. In one embodiment, soluble AIM II polypeptides comprise, or alternatively consist of, all or part of the extracellular domain of an AIM II protein, but lack the transmembrane region that would cause retention of the polypeptide on a cell membrane. Soluble AIM II may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble AIM II protein is capable of being secreted. A heterologous signal peptide can be fused to the N-terminus of the soluble AIM II polypeptide such that the soluble AIM II polypeptide is secreted upon expression.
The invention also provides for AIM II polypeptides, particularly human AIM II polypeptides, which may be employed to treat afflictions such as lymphadenopathy, rheumatoid arthritis, autoimmune disease (e.g., inflammatory autoimmune diseases, myasthenia gravis), graft versus host disease, IgE-mediated allergic reactions, anaphylaxis, adult respiratory distress syndrome, Crohn""s disease, allergic asthma, acute lymphoblastic leukemia (ALL), non-Hodgkin""s lymphoma (NHL), and Graves"" disease. These polypeptides of the invention may also be used to stimulate peripheral tolerance, destroy some transformed cell lines, mediate cell activation and proliferation and are functionally linked as primary mediators of immune regulation and inflammatory response.
The invention further provides compositions comprising, or alternatively consisting of, an AIM II polynucleotide or an AIM II polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise, or alternatively consist of, an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of an AIM II.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by AIM II, which involves contacting cells which express AIM II with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
In another aspect, a screening assay for AIM II agonists and antagonists is provided. The antagonists may be employed to treat, prevent, diagnose, and/or prognose septic shock, inflammation, cerebral malaria, activation of the HIV virus, graft-host rejection, immunodeficiency, bone resorption, and cachexia (wasting or malnutrition). In a preferred embodiment, the AIM II antagonists of the invention (e.g., anti-AIM II antibodies) are used to treat, prevent, diagnose, and/or prognose graft versus host disease.
In a further aspect of the invention, AIM II may be used to treat rheumatoid arthritis (RA) by inhibiting the increase in angiogenesis or increase in endothelial cell proliferation required to sustain an invading pannus in bone and cartilage as is often observed in RA.
In an additional aspect of the invention, AIM II may be used to inhibit or activate a cellular response mediated by a cellular receptor (e.g., LT-xcex2-R, TR2, CD27, and TRANK) by either inhibiting the binding of a ligand to the receptor or by binding to the receptor and activating a receptor mediated cellular response.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of AIM II activity in the body comprising administering to such an individual a composition comprising, or alternatively consisting of, a therapeutically effective amount of an isolated AIM II polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of AIM II activity in the body comprising, administering to such an individual a composition comprising, or alternatively consisting of, a therapeutically effective amount of an AIM II antagonist.